SIMULTANEOUS KINETIC DETERMINATIONS OF LIPASE, CHYMOTRYPSIN, TRYPSIN, ELASTASE, AND AMYLASE ON THE SAME MICROTITER PLATE

被引:58
作者
LAINE, J [1 ]
BEATTIE, M [1 ]
LEBEL, D [1 ]
机构
[1] UNIV SHERBROOKE,DEPT BIOL,CTR RECH MECANISMES SECRET,SHERBROOKE J1K 2R1,QUEBEC,CANADA
关键词
ENZYME ASSAYS; EXOCRINE PANCREAS; GRANULES; MICROMETHODS; SECRETION; ZYMOGENS;
D O I
10.1097/00006676-199305000-00016
中图分类号
R57 [消化系及腹部疾病];
学科分类号
摘要
Micromethods are described to determine in 10 min the activity of the five most common pancreatic zymogens: amylase, lipase, trypsin, chymotrypsin, and elastase. Progress of the reactions is monitored at 405 nm, allowing the kinetic determination of the five enzymes on a single 96-well microtiter plate. Amylase activity is measured by the release of p-nitrophenol from a chemically defined substrate. Linearity of the assay is from 10 to 360 U/L of amylase, and activities as low as 0.4 U/L can be easily measured by extending the period of incubation up to 24 h. Chymotrypsin, trypsin, and elastase activities are monitored by the release of p-nitroanilide from specific substrates, and activities are from 25 to 6,500, 15 to 260, and 20 to 600 U/L, respectively. Finally, lipase is determined by the clearing of a commercially available stabilized emulsion of triolein. The lipase determination can be performed from 90 to 3,600 U/L. When microplate methods were compared with conventional procedures, a perfect correspondence was found between the two types of procedure. Factors necessary to convert microplate results to those of conventional assays are provided. These microassays make possible the rapid and simultaneous determination of the three main types of pancreatic hydrolases (a glycohydrolase, three proteases, and a lipase) with <5 mul of pancreatic juice by kinetic analysis. They could be easily adopted as routine assays in most research laboratories.
引用
收藏
页码:383 / 386
页数:4
相关论文
共 9 条
[1]  
BADENOCH JL, 1989, CLIN CHEM, V35, P645
[2]   AMYLASES, ALPHA AND BETA [J].
BERNFELD, P .
METHODS IN ENZYMOLOGY, 1955, 1 :149-158
[3]   SYNTHESIS AND ANALYTICAL USE OF A HIGHLY SENSITIVE AND CONVENIENT SUBSTRATE OF ELASTASE [J].
BIETH, J ;
SPIESS, B ;
WERMUTH, CG .
BIOCHEMICAL MEDICINE, 1974, 11 (04) :350-357
[4]   SENSITIVE NEW SUBSTRATE FOR CHYMOTRYPSIN [J].
DELMAR, EG ;
LARGMAN, C ;
BRODRICK, JW ;
GEOKAS, MC .
ANALYTICAL BIOCHEMISTRY, 1979, 99 (02) :316-320
[5]   PREPARATION AND PROPERTIES OF 2 NEW CHROMOGENIC SUBSTRATES OF TRYPSIN [J].
ERLANGER, BF ;
COHEN, W ;
KOKOWSKY, N .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1961, 95 (02) :271-&
[6]  
GILLARD BK, 1977, CLIN CHEM, V23, P2279
[7]   A MODIFIED SPECTROPHOTOMETRIC DETERMINATION OF CHYMOTRYPSIN, TRYPSIN, AND THROMBIN [J].
HUMMEL, BCW .
CANADIAN JOURNAL OF BIOCHEMISTRY AND PHYSIOLOGY, 1959, 37 (12) :1393-1399
[8]   THE INTEGRAL AND PERIPHERAL PROTEINS OF THE ZYMOGEN GRANULE MEMBRANE [J].
LEBEL, D ;
BEATTIE, M .
BIOCHIMICA ET BIOPHYSICA ACTA, 1984, 769 (03) :611-621
[9]  
SATVINDER KS, 1990, ANAL BIOCHEM, V188, P188